(PQC2) Niche-responsive RNA editing by ADAR1 in dormant multiple myeloma initiating cell maintenance

 描述（由申请人提供）：尽管有新的可用疗法，包括免疫调节药物，如沙利度胺和来那度胺治疗多发性骨髓瘤（MM），但仍有约20-25%的患者被认为具有治疗失败的高风险。即使在强化治疗方案下，治疗无效病例和疾病复发的出现表明骨髓（BM）内存在能够药物逃逸的休眠骨髓瘤起始群体。这些骨髓瘤起始细胞的定义是缺乏浆细胞标志物CD 138，而它们表达几种造血干细胞（CD 38）和B细胞标志物，如CD 27和CD 19;然而，迄今为止，调节骨髓瘤起始细胞生成和维持的分子和细胞机制知之甚少。最近，我们和其他研究小组表明，NOTCH信号传导和炎症反应性ADAR 1激活都是调节骨髓微环境中恶性干细胞维持的关键事件，其特征在于休眠祖细胞的存活和自我更新以及细胞周期改变增强。在这种情况下，这个中心假设 提出MM小生境来源的促炎信号诱导由腺苷脱氨酶驱动的异常人类特异性RNA编辑，所述腺苷脱氨酶作用于休眠的骨髓瘤起始细胞中的dsRNA-1（ADAR 1），其通过来那度胺抗性而加重。该项目将：1）确定ADAR 1活性是否在骨髓瘤起始细胞中增强，并研究来那度胺治疗对治疗抗性和复发中ADAR 1依赖性RNA编辑的影响; 2）鉴定在骨髓瘤起始细胞中激活ADAR 1依赖性RNA编辑的NOTCH调节的促炎细胞因子;和3）确定是否直接抑制MM起始细胞中的ADAR 1活性，或阻断激活ADAR 1的微环境信号，使骨髓瘤起始细胞对来那度胺敏感并防止骨髓瘤起始细胞维持。这些目标将解决PQC 2：什么分子或细胞事件在治疗后建立肿瘤休眠，什么导致复发？我们将使用流式细胞术和基于荧光RNA探针的策略对骨髓瘤起始细胞功能进行体外和体内测量，以纯化和分析原发性MM细胞成分，并结合基于qPCR的新型诊断性测定以检测内源性RNA编辑，以及基因敲除（CRISPR）或慢病毒shRNA敲减策略以调节NOTCH依赖性ADAR 1激活。荧光泛素化细胞周期指示剂（FUCCI）双顺反子慢病毒报告将有助于在稳健的骨髓基质共培养模型和生物发光人源化MM小鼠模型中研究休眠的活细胞。最终目标是研究小生境依赖性ADAR 1激活作为一种新的机制，驱动休眠骨髓瘤起始细胞的转录组重新编码和分子进化，为靶向治疗奠定基础，并在一系列其他治疗上难以治愈的恶性肿瘤中具有潜在的应用。

 DESCRIPTION (provided by applicant): Despite new available therapies, including immunomodulatory drugs such as thalidomide and lenalidomide in multiple myeloma (MM), about 20-25% of patients are still considered at high risk for treatment failure. The emergence of therapeutically recalcitrant cases and disease relapse even under intensive treatment regimens suggests the existence of a dormant myeloma-initiating population within the bone marrow (BM) that is capable of drug escape. These myeloma-initiating cells are defined by the lack of the plasma cell marker CD138, while they express several hematopoietic stem (CD38) and B cell markers such as CD27 and CD19; however, the molecular and cellular mechanisms that regulate myeloma-initiating cell generation and maintenance are so far poorly understood. Recently, we and other groups showed that both NOTCH signaling and inflammation- responsive ADAR1 activation are crucial events regulating malignant stem cell maintenance in the bone marrow microenvironment, characterized by enhanced survival and self-renewal and cell cycle alterations of dormant progenitor cells. In this context, the central hypothesis of this proposal is that MM niche-derived pro- inflammatory signals induce aberrant human-specific RNA editing driven by adenosine deaminase acting on dsRNA-1 (ADAR1) in dormant myeloma-initiating cells that is accentuated by lenalidomide resistance. This project will: 1) determine whether ADAR1 activity is enhanced in myeloma-initiating cells and investigate the effects of lenalidomide treatment on ADAR1-dependent RNA editing in therapeutic resistance and relapse; 2) identify the NOTCH-regulated pro-inflammatory cytokines that activate ADAR1-dependent RNA editing in myeloma-initiating cells; and 3) determine whether direct inhibition of ADAR1 activity in MM initiating cells, or blocking microenvironmental signals that activate ADAR1, sensitizes myeloma-initiating cells to lenalidomide and prevents myeloma-initiating cell maintenance. These aims will address PQC2: What molecular or cellular events establish tumor dormancy after treatment and what leads to recurrence? We will utilize both in vitro and in vivo measures of myeloma-initiating cell function using multicolor flow cytometry and fluorescent RNA probe-based strategies to purify and profile primary MM cellular constituents, coupled with a novel diagnostic qPCR-based assay to detect endogenous RNA editing, and gene knockout (CRISPR) or lentiviral shRNA- knockdown strategies to modulate NOTCH-dependent ADAR1 activation. A fluorescent ubiquitination cell cycle indicator (FUCCI) bi-cistronic lentiviral reporer will facilitate investigations of dormant live cells, in robust bone marrow stromal co-culture models and bioluminescent humanized MM mouse models. The ultimate goal is to investigate niche-dependent ADAR1 activation as a novel mechanism driving transcriptome recoding and molecular evolution of dormant myeloma-initiating cells, laying the groundwork for targeted therapeutics with potential applications in an array of other therapeutically recalcitrant malignancies.